Antibodies are proteins that exhibit binding specificity to a particular antigen. Native (i.e., naturally occurring or wild-type) antibodies are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. As shown in FIG. 1, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain.
Certain portions of the variable domains differ extensively in sequence among antibodies and are responsible for the binding specificity of each particular antibody to its particular antigen. The constant domains are not involved directly in binding of an antibody to an antigen, but exhibit various effector functions. Depending on the amino acid sequence of the constant region of the heavy chains, antibodies or immunoglobulins can be assigned to different classes. There are five major classes (isotypes) of immunoglobulins in humans: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (subtypes), such as IgG1, IgG2, IgG3, and IgG4 as well as IgA1 and IgA2.
A schematic representation of the native IgG structure is shown in FIG. 1, where the various portions of the native antibody molecule are indicated. The heavy chain constant region includes CH1, the hinge region, CH2, and CH3. Papain digestion of antibodies produces two fragments, Fab and Fc. The Fc fragment consists of CH2, CH3, and part of the hinge region. The crystal structure of the human IgG1 Fc fragment has been determined (Deisenhofer, Biochemistry 20:2361–2370 (1981)). In human IgG molecules, the Fc fragment is generated by papain cleavage of the hinge region N-terminal to Cys 226. Therefore, the human IgG heavy chain Fc region is usually defined as stretching from the amino acid residue at position 226 to the C-terminus (numbered according to the EU index of Kabat, et al., “Sequences of Proteins of Immunological Interest”, 5th ed., National Institutes of Health, Bethesda, Md. (1991); the EU numbering scheme is used hereinafter).
The Fc region is essential to the effector functions of antibodies. The effector functions include initiating complement-dependent cytotoxicity (CDC), initiating phagocytosis and antibody-dependent cell-mediated cytotoxicity (ADCC), and transferring antibodies across cellular barriers by transcytosis. In addition, the Fc region is critical for maintaining the serum half-life of an antibody of class IgG (Ward and Ghetie, Ther. Immunol. 2:77–94 (1995)).
Studies have found that the serum half-life of an IgG antibody is mediated by binding of Fc to the neonatal Fc receptor (FcRn). FcRn is a heterodimer consisting of a transmembrane α chain and a soluble β chain (β2-microglobulin). FcRn shares 22–29% sequence identity with Class I MHC molecules and has a non-functional version of the MHC peptide-binding groove (Simister and Mostov, Nature 337:184–187 (1989)). The α1 and α2 domains of FcRn interact with the CH2 and CH3 domains of the Fc region (Raghavan et al., Immunity 1:303–315 (1994)).
A model has been proposed for how FcRn might regulate the serum half-life of an antibody. As shown in FIG. 2, IgGs are taken up by endothelial cells through non-specific pinocytosis and then enter acidic endosomes. FcRn binds IgG at acidic pH (<6.5) in endosomes and releases IgG at basic pH (>7.4) in the bloodstream. Accordingly, FcRn salvages IgG from a lysosomal degradation pathway. When serum IgG levels decrease, more FcRn molecules are available for IgG binding so that an increased amount of IgG is salvaged. Conversely, if serum IgG levels rise, FcRn becomes saturated, thereby increasing the proportion of pinocytosed IgG that is degraded (Ghetie and Ward, Annu. Rev. Immunol. 18:739–766 (2000)).
Consistent with the above model, the results of numerous studies support a correlation between the affinity for FcRn binding and the serum half-life of an antibody (Ghetie and Ward, ibid.). Significantly, such a correlation has been extended to engineered antibodies with higher affinity for FcRn than their wild-type parent molecules.
Ghetie et al. randomly mutagenized position 252, position 254, and position 256 in a mouse IgG1 Fc-hinge fragment. One mutant showed an affinity three and a half times higher for mouse FcRn and a half-life about 23% or 65% longer in two mouse strains, respectively, as compared to that of the wild-type (Ghetie et al., Nat. Biotechnol. 15:637–640 (1997)).
Shields et al. used alanine scanning mutagenesis to alter residues in the Fc region of a human IgG1 antibody and then assessed the binding to human FcRn. They found several mutants with a higher binding affinity for human FcRn than the wild-type, but did not identify mutations at positions 250, 314, or 428 (Shields et al., J. Biol. Chem. 276:6591–6604 (2001)).
Martin et al. proposed mutagenesis at a number of positions in the human IgG Fc to increase binding to FcRn including, among many others, positions 250, 314, and 428. However, none of the mutants proposed by Martin et al. was constructed or tested for binding to FcRn (Martin et al., Mol. Cell 7:867–877 (2001)).
Dall'Acqua et al. described random mutagenesis and screening of human IgG1 hinge-Fc fragment phage display libraries against mouse FcRn. They disclosed random mutagenesis of positions 428–436 but did not identify mutagenesis at position 428 as having any effect on mouse FcRn binding affinity and stated that the wild-type methionine amino acid at this position is favorable for efficient binding (Dall'Acqua et al., J. Immunol. 169:5171–5180 (2002)).
Kim et al. mutagenized human IgG1 by amino acid substitutions at position 253, position 310, or position 435 of the Fc region. They found that the mutant Fc-hinge fragments have reduced serum half-lives in mice compared to the wild-type IgG1 Fc-hinge fragment, and concluded that Ile253, His310, and His435 play a central role in regulating the serum half-life of IgG (Kim et al., Eur. J. Immunol. 29:2819–2825 (1999)).
Hornick et al. showed that a single amino acid substitution at position 253 in the Fc region of a chimeric human IgG1 antibody accelerates clearance in mice and improves immunoscintigraphy of solid tumors (Hornick et al., J. Nucl. Med. 41:355–362 (2000)).
U.S. Pat. No. 6,165,745 discloses a method of producing an antibody with a decreased biological half-life by introducing a mutation into the DNA segment encoding the antibody. The mutation includes an amino acid substitution at position 253, 310, 311, 433, or 434 of the Fc-hinge domain. The full disclosure of U.S. Pat. No. 6,165,745, as well as the full disclosure of all other U.S. Patent references cited herein, are hereby incorporated by reference.
U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370 disclose the humanization of immunoglobulins.
U.S. Pat. No. 6,277,375 B1 discloses a composition comprising a mutant IgG molecule having an increased serum half-life relative to the wild-type IgG, wherein the mutant IgG molecule comprises the amino acid substitutions: threonine to leucine at position 252, threonine to serine at position 254, or threonine to phenylalanine at position 256. A mutant IgG with an amino acid substitution at position 433, 435, or 436 is also disclosed.
U.S. Patent Application No. 20020098193 A1 and PCT Publication No. WO 97/34621 disclose mutant IgG molecules having increased serum half-lives relative to IgG wherein the mutant IgG molecule has at least one amino acid substitution in the Fc-hinge region. However, no experimental support is provided for mutations at positions 250, 314, or 428.
U.S. Pat. No. 6,528,624 discloses a variant of an antibody comprising a human IgG Fc region, which variant comprises an amino acid substitution at one or more of amino acid positions 270, 322, 326, 327, 329, 331, 333, and 334 of the human IgG Fc region.
PCT Publication No. WO 98/05787 discloses deleting or substituting amino acids at positions 310–331 of the BR96 antibody in order to reduce its induced toxicity, but does not disclose amino acid modifications that result in altered binding to FcRn.
PCT Publication No. WO 00/42072 discloses a polypeptide comprising a variant Fc region with altered FcRn binding affinity, which polypeptide comprises an amino acid modification at any one or more of amino acid positions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305, 307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 386, 388, 400, 413, 415, 424, 433, 434, 435, 436, 439, and 447 of the Fc region, wherein the numbering of the residues in the Fc region is that of the EU index (Kabat et al., op. cit.).
PCT Publication No. WO 02/060919 A2 discloses a modified IgG comprising an IgG constant domain comprising one or more amino acid modifications relative to a wild-type IgG constant domain, wherein the modified IgG has an increased half-life compared to the half-life of an IgG having the wild-type IgG constant domain, and wherein the one or more amino acid modifications are at one or more of positions 251, 253, 255, 285–290, 308–314, 385–389, and 428–435. However, no examples of mutations at positions 314 or 428 with altered binding to FcRn are disclosed.
Martin, W. L. (Doctoral dissertation entitled, “Protein-Protein Recognition: The Neonatal Fc Receptor and Immunoglobulin G,” California Institute of Technology (2001)) proposes theoretical mutations at several Fc positions of the rat gamma-2a constant region, including positions 250 and 428, that may increase FcRn binding affinity. Martin suggests the possibility of substituting, among others, isoleucine for valine at position 250, or substituting phenylalanine for leucine at position 428. Martin does not suggest any substitution for position 314. Martin does not demonstrate increased binding affinity to FcRn for any of these proposed mutations.
The above-referenced publications have not showed that the serum half-life or FcRn binding affinity of an antibody of the IgG class can be altered by the amino acid modifications at position 250, position 314, or position 428 of the Fc region. The present invention used molecular modeling to select Fc residues near the FcRn contact site that might have an effect on binding, but may not be necessary for pH-dependent binding. Amino acid modifications were made at position 250, 314, or 428 of the constant region of an immunoglobulin heavy chain of class IgG. The serum half-lives or FcRn binding affinities of antibodies comprising said modifications were altered and, therefore, were different from those of unmodified antibodies.